Biomimetic hydroxyapatite synthesis

ABSTRACT

A method for preparing nanoscale hydroxyapatite particles by combining an amount of a calcium ion source, which includes calcium acetate, and an amount of a phosphate ion source, wherein the amounts are sufficient to produce nanoscale hydroxyapatite particles and the amounts are combined under ambient conditions to produce the hydroxyapatite particles. Nanoscale hydroxyapatite particles are also presented.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.11/622,927, which was filed on Jan. 12, 2007, which claims priority toU.S. Provisional Application Ser. No. 60/758,207, which was filed onJan. 12, 2006. The foregoing applications are hereby incorporated byreference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of grantDGE033196 awarded by the National Science Foundation.

BACKGROUND OF THE INVENTION

Hydroxyapatite (HAp, chemical formula Ca₁₀(PO₄)₆(OH)₂) has attracted theattention of researchers over the past thirty years as an implantmaterial because of its excellent biocompatibility and bioactivity. HAphas been extensively used in medicine for implant fabrication. It iscommonly the material of choice for the fabrication of dense and porousbioceramics. Its general uses include biocompatible phase-reinforcementin composites, coatings on metal implants and granular fill for directincorporation into human tissue. It has also been extensivelyinvestigated for non-medical applications such as a packingmaterial/support for column chromatography, gas sensors and catalysts,as a host material for lasers, and as a plant growth substrate.

Previously explored methods of hydroxyapatite synthesis for particlesinclude plasma spraying, hydrothermal synthesis, freeze drying, sol-gel,phase transformation, mechanochemical synthesis, chemical precipitation,and precipitation in simulated body fluid (SBF). All of these methodsproduce products with varying levels of purity, size, crystallinity, andyield. Plasma spraying, hydrothermal synthesis, sol-gel, phasetransformation, mechanochemical synthesis, and chemical precipitationrequire elevated temperatures and/or extreme pH values in thefabrication of hydroxyapatite. These conditions can raise importantquestions among biologists when considering the material for in vivoapplications because they are not biomimetic and, in most cases, do notyield biomimetic structures or morphologies. Furthermore, precipitationin simulated body fluid has such a low yield or long reaction time, itis not practical for use in manufacturing implants.

Therefore, a need exists for hydroxyapatite synthesis to take place atroom temperature and optional neutral pH to allow the exploration ofsynthesis with live cells, including those in living organisms.

SUMMARY OF THE INVENTION

There is provided, in accordance with the present invention, a methodfor preparing nanoscale hydroxyapatite particles by combining an amountof a calcium ion source, which includes calcium acetate, and an amountof a phosphate ion source, wherein the amounts are sufficient to producenanoscale hydroxyapatite particles and the amounts are combined underessentially ambient conditions to produce the hydroxyapatite particles.

One embodiment includes a stable colloidal suspension of nanoscalehydroxyapatite particles suspended in a biocompatible ionic solutionprepared by the method of the present invention, wherein the ionicsolution includes physiological concentrations of phosphate and acetateanions and sodium or potassium cations.

Also provided are powdered hydroxyapatite particles having a BET surfacearea between about 200 and about 3000 m²/g and a particle size betweenabout 1 nm and about 9 nm.

In one embodiment, a stable colloidal suspension of the hydroxyapatiteparticles suspended in a biocompatible ionic solution is provided.

Also provided is a kit for use in preparing nanoscale hydroxyapatiteparticles, wherein the kit includes (a) an amount of a calcium ionsource, which includes calcium acetate, and (b) an amount of a phosphateion source, wherein the amounts are sufficient to produce nanoscalehydroxyapatite particles when combined under ambient conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 a-d are transmission electron microscopy (TEM) images ofparticles prepared according to the method of Example 2;

FIGS. 2 a-b are TEM images of particles prepared according to the methodof Example 3;

FIG. 2 c is a TEM image of particles prepared according to the method ofExample 3 at a higher magnification than the images of FIGS. 2 a-b;

FIG. 2 d is a TEM image of particles prepared according to the method ofExample 3 showing a particle size distribution of +/−10 nm;

FIG. 2 e is a high resolution transmission electron microscopy (HRTEM)image of particles prepared according to the method of Example 3;

FIG. 3 a is an XRD spectrum corresponding to HAp particles preparedaccording to a method of Example 1;

FIG. 3 b is an XRD spectrum corresponding to HAp particles preparedaccording to the method of Example 2;

FIG. 3 c is an XRD spectrum corresponding to HAp particles preparedaccording to a method of Example 1, wherein the reactant concentrationswere halved; and

FIG. 3 d is an XRD spectrum corresponding to HAp particles preparedaccording to a method of Example 1, wherein the particles were heattreated at 900° C. for 2 hours.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to methods for preparing nanoscale HApparticles and the HAp particles prepared therewith. Kits for use inpreparing the particles and colloids containing the particles are alsopresented.

Hydroxyapatite has reported uses for biomedical, chromatographic, andpiezoelectric applications and has been synthesized by varioustechniques. However, reaction conditions for the preparation of HAp suchas high temperatures, high pressures and extreme pH values, as well aslow yield, vigorous washing requirements, and long reaction times limitbiological applications.

The methods of the present invention permit the formation under mildreaction conditions of HAp under conditions suitable for the above uses,especially biological use.

The method involves combining an amount of a calcium ion source, whichincludes calcium acetate, and an amount of a phosphate ion source,wherein the amounts are sufficient to produce nanoscale HAp particlesand the amounts are combined under essentially ambient conditions toproduce the HAp particles.

Suitable phosphate ion sources include, but are not limited to, one ormore of potassium or sodium orthophosphate; orthophosphoric acid; GroupI phosphates, preferably monobasic, dibasic, or tribasic potassium orsodium phosphate; magnesium phosphate; ammonium phosphate; and the like.Potassium or sodium orthophosphate is preferred. In addition to calciumacetate, the calcium ion source may include one or more of calciumhydroxide, calcium oxalate, calcium acetate, calcium nitrate, calciumphosphate, calcium carbonate, calcium fluoride, and calcium chloride.Calcium acetate alone is preferred.

The calcium ion source, the phosphate ion source, or both are insolution prior to combining the sources. Preferably, the solutioncontains one or more of water, buffer, solvent, simulated body fluid, orfortified cell medium with or without serum. Suitable buffers include,but are not limited to,N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (HEPES),2-(bis(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diol(BIS-TRIS), 3-(N-Morpholino)-propanesulfonic acid (MOPS),N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES),N-(2-Acetamido)iminodiacetic Acid (ADA),N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic Acid (BES),3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO),4-(N-morpholino)butanesulfonic acid (MOBS),3-[N-morpholino]-2-hydroxypropanesulfonic acid (MOPSO),piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES),3-[N-Tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid(TAPSO), N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES),and acetic acid. A preferred buffer is acetic acid.

If a particular ion source is not in solution, the source is in a solidphase.

An optional step includes agitating the combination until HAp is formed.Agitating the combination accelerates the formation of hydroxyapatite.As used herein, the term “agitate” refers to mechanical movement, forexample, vibrating, vortexing, swirling, shaking, ultrasonicating,stirring, or the like that causes mixing. Mechanical movements includemovements performed by hand.

When the precursor composition is completely mixed, an extremely viscousgel is formed. This thixotropic material exhibits shear thinning afterfurther intense agitation and returns to a milky solution. Optionally,this solution is allowed to age for a period between 2 minutes and 10days. During the aging process, additional agitation may or may not beapplied to continue mixing. The aging time allows Ostwald ripening ofthe particles, therefore, the particles aged longer exhibit a largerparticle size.

Essentially ambient conditions are employed. A preferred temperaturerange is between −10° C. and 45° C. At room temperature, HAp particlesare typically produced within 1 minute to an hour. Combining the sourceswhile heating will speed up the rate of reaction to more quickly produceHAp, while combining the ion sources while cooling will decrease therate at which HAp particles form.

During the course of the reaction, a pH swing may occur, which is variedwith the calcium to phosphate stoichiometry. A preferred embodiment, inwhich the calcium to phosphate ratio is about 1.67, exhibits a pH swingof 12 down to 7 over the time of a 4 hour reaction. The pH of the gelphase is 12; just after the shear thinning and return to solution, thepH is 10; after 3 hours of reaction, the pH is 8; and after 4 hours ofreaction, the pH returns to neutral. The time to neutral, however, alsodepends on the employment or omission of agitation throughout thereaction that may enhance kinetics and diffusion of the ions in theformation of hydroxyapatite.

The employment of a buffer as the reaction medium moderates the pHchange, which affects the product formed. Hydroxyapatite is formed, butsecondary phases of calcium phosphate and calcium carbonate may beadditionally formed, but can be remedied through process variations, forexample, bubbling with nitrogen, addition of chelating agents, or use ofadditional pH adjustments or buffers.

Another optional step includes adding one or more dopant ions suitablefor substitution into the HAp lattice. Such ions are readilydeterminable by one of skill in the art. Suitable ions include, but arenot limited to, magnesium, fluorine, chlorine, potassium, iron,carbonate, sodium, and the like. The HAp particles of the presentinvention can also be doped with ions of one or more rare earthelements.

Following the aging process, a washing step can be performed. This stepincludes, for example, filtration, centrifuging, and/or liquidreplacement. Centrifuging or liquid replacement are preferred. Minimalwashing cycles are needed because of the non-toxic nature of the ionsleft in solution. In one embodiment, the citrate wash disclosed in U.S.Pat. No. 6,921,544, the contents of which are incorporated herein byreference in their entirety, is used to remove at least a portion of anamorphous phase if the amorphous phase is considered an undesiredimpurity.

To produce solid hydroxyapatite, the solution is dried. Suitable dryingtechniques are readily determinable by those of skill in the art.Preferred drying techniques include evaporative and sublimation-baseddrying methods, for example, oven drying and freeze drying.

The methods according to the present invention can take place in anysuitable reaction system. An exemplary system includes a flow reactorfor continuous production of hydroxyapatite.

Powdered HAp particles produced according to the methods of the presentinvention are also presented. The HAp particles have a BET surface areabetween about 200 and about 3000 m²/g and a particle size between about1 nm and about 9 nm. In one embodiment, the particles have a dispersedparticle size between about 1 and about 9 nm. The term “dispersed” isdefined herein in the context of colloidal chemistry where the particlesmaintain an inter-particle spacing such that no physical contact is madebetween the particles. The term “aggregated” is defined herein asadjacent particles having no inter-particle spacing and making contactwith the nearest neighboring particles. In another embodiment, theparticles are aggregated and have an aggregated particle size equal toor greater than about 2 nm, preferably between about 2 nm and about 5mm.

The composition of the powdered HAp particles is stoichiometric ornon-stoichiometric with respect to calcium and phosphate. For example,the XRD diffraction pattern of FIG. 3 d represents the results of astandard test for stoichiometry. FIG. 3 d shows the presence of peakscorresponding to tricalcium phosphate (TCP) after the sample was heattreated at 900° C. for 2 hours. The presence of TCP indicates anon-stoichiometric composition and/or an amorphous phase. In oneembodiment, at least a portion of the composition includes an amorphousphase. In one embodiment, the ratio of calcium to phosphate in the HApparticles is between 1.25 and 2.5.

The HAp particles exhibit a spherical or non-spherical morphology. Theterm “spherical” is used herein to mean equiaxed particles having eithera primary or secondary particle structure.

Given that hydroxyapatite has no toxicity and its components are lowcost, such a technology presents great promise for a range ofapplications.

HAp particles having the size distribution of the present invention areeffective in drug delivery because they are more capable of penetratingthe cellular wall and carry a much higher surface area for adsorption ofdrug molecules. The range also allows the particles to be usedintravenously as a drug therapy or for transdermal drug delivery. Thesize range is also important in biomaterial applications because it isclose to what is seen naturally in the body. Being smaller, it will alsobe more readily processable by cells and tissues for regeneration andresorption.

Devices based on hydroxyapatite are typically in the form ofpolycrystalline ceramics, polymer-ceramic composites, or films on ametallic surface such as titanium. The powders produced in thisinvention can be used in conventional processes to make all three formsof materials, using conventional methods such as solid state sinteringfor polycrystalline ceramics, polymer-melt processing forpolymer-ceramic composites and plasma spraying for hydroxyapatite-coatedtitanium metal. The particles of this invention can be grown directlyonto the metal surfaces without the need for any high temperatureprocessing.

The hydroxyapatite of the present invention is also useful in thepreparation of compounds for use as granular fill for directincorporation into the hard tissues of humans or other animals, and asbone implantable materials. The present invention thus includes granularfill compounds, bone implant materials, tooth filling compounds, bonecements and dentifrices containing the hydroxyapatite of the presentinvention. The products are formulated and prepared by substituting thehydroxyapatite of the present invention for hydroxyapatite inconventional hydroxyapatite-based products. The compounds may beprepared from metallic and polymeric hydroxyapatite composites.

Suitable polymers include polysaccharides, poly(alkylene oxides),polyarylates, for example those disclosed in U.S. Pat. No. 5,216,115,block co-polymers of poly(alkylene oxides) with polycarbonates andpolyarylates, for example those disclosed in U.S. Pat. No. 5,658,995,polycarbonates and polyarylates, for example those disclosed in U.S.Pat. No. 5,670,602, free acid polycarbonates and polyarylates, forexample those disclosed in U.S. Pat. No. 6,120,491, polyamide carbonatesand polyester amides of hydroxy acids, for example those disclosed inU.S. Pat. No. 6,284,862, polymers of L-tyrosine derived diphenolcompounds, including polythiocarbonates and polyethers, for examplethose disclosed in U.S. Pat. No. RE37,795, strictly alternatingpoly(alkylene oxide) ethers, for example those disclosed in U.S. Pat.No. 6,602,497, polymers listed on the United States FDA “EAFUS” list,including polyacrylamide, polyacrylamide resin, modified poly(acrylicacid-co-hypophosphite), sodium salt polyacrylic acid, sodium saltpoly(alkyl(C16-22) acrylate), polydextrose,poly(divinylbenzene-co-ethylstyrene),poly(divinylbenzene-co-trimethyl(vinylbenzyl)ammonium chloride),polyethylene (m.w. 2,00-21,000), polyethylene glycol, polyethyleneglycol (400) dioleate, polyethylene (oxidized), polyethyleneiminereaction product with 1,2-dichloroethane, polyglycerol esters of fattyacids, polyglyceryl phthalate ester of coconut oil fatty acids,polyisobutylene (min. m.w. 37,000), polylimonene, polymaleic acid,polymaleic acid, sodium salt, poly(maleic anhydride), sodium salt,polyoxyethylene dioleate, polyoxyethylene (600) dioleate,polyoxyethylene (600) mono-rici noleate, polyoxyethylene 40monostearate, polypropylene glycol (m.w. 1,200-3,000), polysorbate 20,polysorbate 60, polysorbate 65, polysorbate 80, polystyrene,cross-linked, chloromethylated, then aminated with trimethylamine,dimethylamine, diethylenetriamine, or triethanolamine, polyvinylacetate, polyvinyl alcohol, polyvinyl polypyrrolidone, andpolyvinylpyrrolidone, and polymers listed in U.S. Pat. No. 7,112,417,the disclosures of all of which are incorporated herein by reference intheir entirety.

Also presented is a kit for use in preparing nanoscale HAp particles ofthe present invention. The kit includes (a) an amount of a calcium ionsource comprising calcium acetate and (b) an amount of a phosphate ionsource, wherein the amounts are sufficient to produce nanoscale HApparticles when combined under ambient conditions. The kit can be used toprepare HAp particles prior to the introduction of the particles into apatient. Alternatively, the kit can be used to combine components (a)and (b) in a patient in need thereof for the preparation, and subsequentdeposit, of HAp particles in vivo. The two ion sources are provided inseparate containers. Other components may be present depending upon theintended therapeutic use.

Yet another aspect of the present invention includes a stable colloidalsuspension with nanoscale HAp particles suspended in a biocompatibleionic solution prepared according to the methods of the presentinvention, wherein the ionic solution includes physiologicalconcentrations of phosphate and acetate anions and sodium or potassiumcations. Also presented is a stable colloidal suspension with the HApparticles of the present invention suspended in a biocompatible ionicsolution.

A suitable ionic solution is readily determinable by one of skill in theart. In one embodiment, the ionic solution includes the mother liquorfrom which the hydroxyapatite particles were produced. The mother liquorcan be formulated to produce an ionic buffer upon HAp formation. Forexample, the mother liquor could form a phosphate buffered saline (PBS)solution upon formation of HAp. In another embodiment, the ionicsolution includes a solution prepared independently of thehydroxyapatite particles, for example, one of the aforementionedbuffers.

The following non-limiting examples set forth herein below illustratecertain aspects of the invention.

EXAMPLES Example 1 Room Temperature Crystallization of Hydroxyapatite inWater

Calcium acetate hydrate (99% Acros Organics, Belgium, CAS #114460-21-8)and potassium orthophosphate hydrate (Acros Organics, Belgium, CAS#27176-10-9) were used as reactants for the synthesis of hydroxyapatite.First, a 1.0 molal calcium acetate hydrate solution was made usingdistilled, deionized water. Then, a 0.6 molal solution of potassiumorthophosphate hydrate was made using distilled, deionized water. Equalvolumes of each were then measured out for the reaction to create acalcium to phosphate ratio of 1.67 (final concentrations of ions if theywere to remain in solution would be 0.5 m/0.3 m). A 100 mL reactionrequired 50 mL of the calcium solution to be measured and poured into abeaker and 50 mL of the phosphate solution to be added. Agitation viavortexing was then performed until and through the gelation stage. Oncethe gel returned to solution, the slurry was then allowed to age for 2minutes. The concentrate was then dried in an oven at 70° C. for 24hours and desiccated until use or immediately atomized onto aTransmission Electron Microscopy (TEM) grid for characterization.

X-Ray diffraction (XRD) confirmed the phase formed was hydroxyapatitewith no detectable secondary phases or peaks present. FIG. 3 a is an XRDdiffraction pattern corresponding the HAp particles prepared accordingto this method, which were washed followed by drying in a 70° C. oven.The broadness of the peaks in FIG. 3 a indicates nanoscale particles.Furthermore, the peaks match the standard reference peaks forhydroxyapatite. Helium pycnometry confirms an average particulatedensity of 2.4 g/cm³.

FIG. 3 c is an XRD diffraction pattern corresponding to HAp particlesprepared according to this method, wherein the reactant concentrationswere halved. FIG. 3 c also confirms the presence of HAp particles.

Example 2 Room Temperature Crystallization of Hydroxyapatite in Water

Hydroxyapatite was prepared according to the method of Example 1.However, a 2.0 molal calcium acetate hydrate solution and a 1.2 molalsolution of potassium orthophosphate hydrate were used. The finalconcentrations of ions if they were to remain in solution after mixingwould be 1.0 m/0.6 m.

TEM images of the resulting hydroxyapatite particles are shown in FIGS.1 a-d. These images show lattice fringes indicating crystallinity of theparticles and also show relatively uniform particle size and morphology.

The XRD diffraction pattern is shown in FIG. 3 b, which also confirmsthe presence of HAp particles.

Example 3 Room Temperature Crystallization of Hydroxyapatite in a SelfBuffering Solution

Calcium acetate hydrate and potassium orthophosphate hydrate solutionswere prepared as described in Example 1. The potassium orthophosphatehydrate solution was divided in half and acetic acid was added to one ofthe two portions until the pH of the solution was 7.4 (volume depends ontotal solution volume, for example 500 mL solution needs about 23 mL ofglacial acetic acid). Proportional amounts of each of the threesolutions were then measured out to create a calcium to phosphate ratioof 1.67 and pH of 7.4 (final concentrations of ions if they were toremain in solution would be 0.5 m/0.3 m). A 20 mL reaction required 10mL of the calcium solution to be measured and poured into a beaker and8.5 mL of the phosphate solution (unadjusted) to be added to the calciumsolution followed by 1.5 mL of the pH adjusted solution. Agitation viastirring with a glass rod was then performed until the solution appearedcompletely mixed and white (a gelation is not seen). The slurry was notaged. The concentrate or solution was then dried in an oven at 70° C.for 24 hours and desiccated until use or immediately atomized onto a TEMgrid for characterization.

X-Ray diffraction confirmed the phase formed was hydroxyapatite with nodetectable secondary phases or peaks present. An average density of 2.2g/cm³ was determined via Helium pycnometry.

TEM images of the resulting hydroxyapatite particles are shown in FIGS.2 a-e. These images show lattice fringes indicating crystallinity of theparticles and also show relatively uniform particle size and morphology.Dispersed particles having a particle size less than 10 nm are shown.

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thespirit and script of the invention, and all such variations are intendedto be included within the scope of the following claims.

What is claimed is:
 1. A method for preparing nanoscale hydroxyapatiteparticles comprising combining an amount of a calcium ion sourcecomprising calcium acetate and an amount of a phosphate ion source,wherein said amounts are sufficient to produce nanoscale hydroxyapatiteparticles consisting essentially of crystals characterized by a dominantlattice spacing of about 0.28 nm and said amounts are combined at atemperature between −10° C. and 45° C. to produce said hydroxyapatiteparticles by direct crystallization.
 2. The method of claim 1, whereinsaid phosphate ion source is selected from the group consisting ofpotassium orthophosphate, sodium orthophosphate, orthophosphoric acid,Group I phosphates, magnesium phosphate, ammonium phosphate, and acombination of two or more thereof.
 3. The method of claim 2, whereinsaid Group I phosphates are selected from the group consisting ofmonobasic sodium phosphate, dibasic sodium phosphate, tribasic sodiumphosphate, monobasic potassium phosphate, dibasic potassium phosphate,and tribasic potassium phosphate.
 4. The method of claim 1, wherein saidcalcium ion source further comprises calcium hydroxide, calcium oxalate,calcium nitrate, calcium phosphate, calcium carbonate, calcium fluoride,calcium chloride, or a combination of two or more thereof.
 5. The methodof claim 1 further comprising agitating the combination untilhydroxyapatite is formed.
 6. The method of claim 1, further comprisingdecreasing the temperature of the combined ion sources to a temperaturenot less than −10° C. until hydroxyapatite is formed.
 7. The method ofclaim 1, further comprising increasing the temperature of the combinedion sources to a temperature not greater than 45° C. untilhydroxyapatite is formed.
 8. The method of claim 1, further comprisingadding a buffer solution to the combination.
 9. The method of claim 1,wherein the production of said hydroxyapatite particles is stoppedbefore the hexagonal structure is completely formed.